The goal of the proposed study is to identify and determine the role of microRNAs (miRNAs) and their targets in modulating innate immunity signaling and gut epithelial homeostasis in response to pathogen infection in Drosophila, in order to provide new insights to better understand their evolutionarily conserved mammalian counterparts. In response to bacterial or fungal infection via septic injury, the immune deficiency or Toll signaling cascades, respectively, are activated, culminating in the production of a battery of potent antimicrobial peptides (AMPs). AMPs are secreted into the hemolymph and function as systemic defense agents to clear invading pathogens. In addition, upon encountering pathogens via natural routes of infection such as oral ingestion, a local immune response is mounted in the Drosophila gut, leading to the synthesis of microbicidal reactive oxygen species and AMPs. The molecular mechanism underlying innate immunity signaling is remarkably conserved between flies and mammals. While effective control of invading pathogens depends on rapid and robust induction of immunity signaling pathways, prolonged or aberrant activation, either systemically or locally, can lead to pathological conditions in humans. Therefore, both the magnitude and duration of innate immunity signaling need to be tightly regulated. In addition, pathogen infection of the fly gastrointestinal tract triggers a homeostatic response by stimulating intestinal stem cell proliferation and differentiation to repair damaged tissues. Dysregulation of this homeostatic program can cause a dysplasia phenotype that remarkably resembles colorectal cancer in humans. miRNAs are a class of endogenous regulatory small RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. We hypothesize that select miRNAs play a key role in regulating innate immunity signaling pathways and gut homeostasis in response to pathogen infection in Drosophila. Here we propose to identify and elucidate the role of these miRNAs and their targets in modulating these biological processes, aiming to both integrate miRNAs into the Drosophila innate immunity signaling and tissue homeostasis network and to exploit miRNAs as tools to uncover from miRNA target genes novel factors that regulate these processes. Completion of the proposed study will establish miRNAs as modulators of innate immunity signaling and gut homeostasis in response to pathogen infection in Drosophila. This will advance our understanding of their conserved mammalian counterparts, provide new insights to the mechanisms underlying human pathological conditions such as autoimmunity, inflammatory diseases and cancer, and may facilitate the development of therapeutic agents to treat these devastating disorders.